DNAs and proteins or peptides specific to bacteria of the species Neisseria meningitidis, processes for obtaining them and their biological uses

ABSTRACT

The DNA of the invention are characterised in that they concern the whole or part of genes, with their reading frame, to be found in  Neisseria meningitidis , but not in  Neisseria gonorrhoeae , or in  Neisseria lactamica  except the genes involved in the biosynthesis of the polysaccharide capsule, frp A, frp C, opc, por A, rotamase the sequence IC1106, IgA protease, pilline, pilC, transferrin binding proteins and opacity proteins. The invention also concerns the polypeptides corresponding to these DNA and the antibodies directed against these polypeptides. It is applicable in the prevention and the detection of meningococcus induced infections and meningitis.

The present application is a continuation of application Ser. No.09/214,759, filed Apr. 22, 1999 now abandoned, which is a 371application of PCT/FR97/01295, filed Jul. 11, 1997, and claims benefitof FR 96 08768, filed Jul. 12, 1996.

DNAs and proteins or peptides specific to bacteria of the speciesNeisseria meningitidis, processes for obtaining them and theirbiological uses.

The invention relates to DNAs and to proteins and peptides which arespecific to bacteria of the species Neisseria meningitidis (abbreviatedbelow to Nm), to the process for obtaining them and to their biologicaluses, in particular for the prevention and detection of meningococcalinfections and meningitis.

It is known that Nm is one of the main agents of cerebrospinalmeningitis.

Studies conducted in the United States have shown that 5 to 10% of thepopulation are asymptomatic carriers of the Nm strain(s). Thetransmission factors of Nm are poorly known. For a proportion of personsinfected, Nm penetrates the bloodstream, where it can causemeningococcaemia and/or progress to the cerebrospinal stream, to causemeningitis. Without fast antibiotic treatment, the infection can developlike lightning and become fatal.

Compared with other pathogens, Nm has the characteristic of being ableto cross the haemato-encephalic barrier to colonize the meninges. Thestudy of the pathogenicity of Nm is therefore important not only in thecontext of meningitis, but also in the context of any disease whichaffects the brain.

The benefit of having available tools specific to this species ofbacteria for the uses envisaged above is therefore understood.

Genetically, Nm is very close to bacteria of the species Neisseriagonorrhoeae (abbreviated to Ng below) and of the species Neisserialactamica (abbreviated to Nl below). However, their pathogenicity isvery different.

Nm colonizes the nasopharynx, and then crosses the pharyngeal epitheliumto invade the submucous space, thus being responsible for septicaemiaand meningitis.

Ng is especially responsible for infections located in the genitourinarytract. It colonizes the genital mucosa, and then crosses the epithelium,subsequently invading the subepithelium, where it multiplies and isresponsible for a severe inflammatory reaction. Disseminated gonococcalinfections are possible, but remain rare and are the result of only somestrains.

As regards Nl, it is considered that this is a non-pathogenic strain,since it is not responsible for a localized or general invasion.

A first consideration thus led to taking into account the fact that Nmand Ng, while being bacteria very close to one another, have differentpathogenic potencies.

Since the genome of these bacteria has a high homology, only limitedparts of the genome of Nm and Ng must code for specific virulencefactors responsible for their pathogenesis.

It is clear that Nm has, compared with Ng, DNA sequences which arespecific to it and which must be involved in the expression of itsspecific pathogenic potency.

The species Nm is subdivided into serogroups based on the nature of thecapsular polysaccharides.

At least 13 serogroups have been defined, among which serogroups A, Band C are responsible for about 90% of meningitis cases. Groups A and Care found in epidemic forms of the disease. Group B is the serogroupgenerally isolated the most in Europe and the United States.

The capsule, which is present in Nm and absent from Ng, has served asthe basis for formulating meningococcal antimeningitis vaccines.

The polysaccharides of the Nm capsule have been used to formulate avaccine which has proved to be effective in preventing in adults themeningitis caused by meningococci of serogroups A, C, W135 and Y.

However, the polysaccharide of Nm group C has proved to be weaklyimmunogenic in children of less than two years, while the polysaccharideof Nm group B is non-immunogenic in man and shares epitopes withadhesion glycoproteins present in human neuronal cells.

There is therefore no universal vaccine capable of preventing infectionscaused by all the serogroups of the meningococci and capable ofresponding to the intrinsic antigenic variability of bacterial pathogensin general and Nm in particular.

Because of the cross-reactivity of the Nm group B polysaccharide withhuman antigens, the multiplicity of the serogroups and the antigenicvariability of Nm, the strategies proposed to date cannot lead to avaccine which is effective in all situations.

Research is therefore concentrated on study of the characteristicelements responsible for the specificity of the meningococcalpathogenesis.

The majority of genes which have been studied in either of the twobacteria Nm or Ng have their homologue in the second bacterium.

In the same way, the majority of virulence factors identified to date inNm have a counterpart in Ng, that is to say pilin, the PilC proteins,the opacity proteins and the receptors of lactoferrin and transferrin.

The specific attributes of meningococci characterized in the prior artare the capsule, the Frp proteins analogous to RTX toxins, Opc proteinsof the external member, glutathione peroxidase, the porin PorA and therotamase gene.

Among these, only the capsule is invariably present in the virulentstrains of Nm. However, several extracellular pathogens have a capsulewithout nevertheless crossing the haemato-encephalic barrier.

Attributes which have not yet been identified must therefore beresponsible for the specificity of the meningococcal pathogenesis. Theseattributes are probably coded by DNA sequences present among themeningococci but absent from the gonococci.

The inventors have developed a new approach based on subtractiveisolation of Nm-specific genes, which genes must be linked to thespecific pathogenesis of Nm, and more particularly to crossing of thehaemato-encephalic barrier.

The subtractive method developed in the prior art has resulted in theproduction of epidemological [sic] markers for some Nm isolates. Thesemarkers are of limited use: they do not cover all the serogroups of theNm species.

In contrast to these studies, the work of the inventors has led, byconfronting Nm with the entire Ng chromosome sheared in a random manner,to the development of a means for cloning all the DNAs present in Nm andabsent from Ng, thus providing tools of high specificity with respect toNm, and thus enabling the genetic variability of the species to beresponded to for the first time.

The terms “present” and absent” used in the description and claims inrelation to the DNAs of a strain or their expression products areinterpreted on the basis of identical hybridization conditions (16 h at65° C., with NaPO₄ 0.5 M, pH 7.2; EDTA-Na 0.001 M, 1%, 1% bovine serumalbumin and 7% sodium dodecylsulphate) using the same probe and the samelabelling intensity of the probe, the same amount of chromosomal DNA andthe same comparison element (chromosomal DNA of the homologous strain).

It is therefore considered that the DNA is present if the signalobtained with the probe is practically the same as that obtained withthe reference strain.

Conversely, it is considered that the DNA is absent if this signalappears very weak.

A second consideration of the pathogenicities of Nm and Ng leads totaking into account their common capacity for colonization andpenetration of the mucosa, and then invasion of the subepithelial spaceof the latter. It is highly probable that this process involvesvirulence factors common to the two pathogens. In this respect, it isknown that a certain number of virulence factors have already beenidentified in Nm and in Ng, such as the pili proteins, PilC, the opacityproteins, the IgA proteases, the proteins for binding to transferrin andto lactoferrin, and the lipooligosaccharides.

The approach of the inventors is thus extended to investigation of theNm regions which are specific to Nm and Ng but absent from thenon-pathogenic species Nl, and in a general manner to investigation ofthe chromosomal regions of the DNAs and their expression productsspecific to a given species by the means developed in accordance withthe invention.

The object of the invention is thus to provide DNAs of Nm specific toits pathogenic potency and means for obtaining them, in particular byformulating banks formed exclusively from these Nm-specific DNAs.

It also provides the products derived from these DNA sequences.

The invention also relates to the utilization of specific and exhaustivecharacteristics of these banks to formulate tools which can be used, inparticular, in diagnostics, treatment and prevention.

The DNAs of the invention are characterized in that they are in all orpart genes, with their reading frame, present in Neisseria meningitidis,but absent either from Neisseria gonorrhoeae and from Neisserialactamica, with the exception genes involved in the biosynthesis of thepolysaccharide capsule, frpA, frpC, opc, por A, rotamase, the sequenceIS1106 (Accession No. Z 11857 in the EMBL/GenBank/DDBJ NucleotideSequence Data Libraries; see Knight et al., 1992, Molecular Microbiology6(11): 1565-1573), IgA proteases, pilin, pilC, proteins which bindtransferrin and opacity proteins.

As stated above, the terms “present” and “absent” are interpreted on thebasis of the hybridization conditions used in the Southern blottingdescribed in the examples and referred to above.

It can be seen that these DNAs include variants where these express afunction intrinsic to the Nm species, more particularly a phenotypefound solely in Nm or in common exclusively with Ng.

According to a main aspect, these DNAs are specific to the pathogenicityof Neisseria meningitidis, in spite of the genetic variability of thisspecies.

According to an embodiment of the invention, the said DNAs are specificto Nm, in contrast to Ng.

More particularly, the Nm-specific DNAs are absent from Neisserialactamica and from Neisseria cinerea.

Surprisingly, the majority of genetic differences between the strains ofmeningococci and those of gonococci appear grouped in distinct regions,which are said to correspond to the pathogenicity islets describedpreviously for E. coli and Y. pestis.

In a preferred embodiment of the invention, these DNA are thus alsocharacterized in that they comprise one or more sequence(s) present onthe chromosome of Neisseria meningitidis Z2491 between tufA and pilT, orregion 1 of the chromosome, and/or the sequence(s) capable ofhybridizing with the above sequence(s), with the proviso of beingspecific to Neisseria meningitidis.

“Specific” in the description and the claims means the nucleotidesequences which hybridize only with those of Nm under the hybridizationconditions given in the examples and referred to above.

In this respect, it can be seen that, in a general manner, when “all orpart” of a sequence is referred to in the description and claims, thisexpression must be interpreted with respect to the specificity definedabove.

Furthermore, all or part of a peptide or a fragment of a peptide or anantibody means a product having the biological properties respectivelyof the natural peptide or the antibody formed against the peptide.

Genes of the Neisseria meningitidis capsule are grouped in region 1.

DNAs of this type have a sequence corresponding in all or part to SEQ IDNo. 9, 13, 22 or 30, and/or to any sequence located at more or less 20kb from these SEQ ID on the chromosome of an Nm strain, and/or have asequence which is capable of hybridizing with at least a fragment of anyone of these sequences.

In another preferred embodiment of the invention, these DNA are alsocharacterized in that they are made up of one or more sequence(s)present on the chromosome of Neisseria meningitidis Z2491 between pilQand λ740, or region 2 of the chromosome, and/or the sequences(s) capableof hybridizing with the above sequence(s), with the proviso of beingspecific to Neisseria meningitidis.

DNAs according to this embodiment have a sequence corresponding in allor part to SEQ ID No. 1, 2, 4, 6, 7, 10, 15, 31 or 34, and/or to anysequence located at more or less 20 kb from these SEQ ID on thechromosome of an Nm strain, and/or have a sequence which is capable ofhybridizing with at least a fragment of any one of these sequences.

The invention especially provides all or part of the DNA sequence SEQ IDNo. 36 of 15,620 bp, and the sequences corresponding to the open readingframes SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44 and SEQ ID No.45.

In yet another preferred embodiment of the invention, these DNAs arealso characterized in that they are made up of one or more sequence(s)present on the chromosome of Neisseria meningitidis Z2491 between argFand opaB, or region 3 of the chromosome, and/or the sequence(s) capableof hybridizing with the above sequence(s), with the proviso of beingspecific to Neisseria meningitidis.

DNAs according to this embodiment are characterized in that they have asequence corresponding in all or part to SEQ ID No. 8, 21, 23, 25, 26,28, 29, 32 or 35, and/or to any sequence located at more or less 20 kbfrom these SEQ ID on the chromosome of an Nm strain, and/or have asequence which is capable of hybridizing with at least a fragment of anyone of these sequences.

Regions 1, 2 and 3 identified above have a high proportion of sequencesspecific to Neisseria meningitidis and also fall within the context ofthe invention.

Other DNAs representative of the specificity with respect to Neisseriameningitidis have one or more sequences which is/are present on thechromosome of Neisseria meningitidis Z2491 but are not part of regions1, 2 and 3 defined above.

Such DNAs comprise one or more sequence(s) corresponding in all or partto SEQ ID No. 3, 5, 11, 12, 14, 16, 18, 19, 20, 24, 27 or 33, and/or toany sequence located at more or less 20 kb from these SEQ ID on thechromosome of an Nm strain, and/or have a sequence capable ofhybridizing with such sequences.

Taking into account the uses envisaged in particular, the invention morespecifically relates to the above DNAs involved in the pathogenesis ofthe bacterial organism.

In particular, it provides the DNAs corresponding to at least one of thecharacterizations given above and coding for a protein exported beyondthe cytoplasmic membrane, and/or of which all or part of their sequencecorresponds to the conserved region of the said DNAs.

According to another embodiment of the invention, the DNAs are thuscommon with those of Ng, but are absent from those of Nl.

These are more specifically the DNAs which are present on region 4 (argJ to reg F) or on region 5 (lambda 375 marker to pen A) on thechromosome of Nm Z2491 and/or are capable of hybridizing with the saidDNAs present, with the proviso of being specific to Nm and Ng, incontrast to Nl.

“Specific to Nm and Ng in contrast to Nl” means the DNAs which hybridizewith the DNAs of Nm and Ng under the hybridization conditions of theexamples (see example 4 in particular).

The DNAs of regions 4 and 5 and those capable of hybridizing with theseDNAs, with the proviso of expressing the intrinsic functions of Nm, havethe advantage of intervening in a significant manner in the virulence ofNm, being involved in the stage of initial colonization and penetrationand in the septicaemic dissemination.

According to other embodiments, the invention provides transfer andexpression vectors, such as plasmids, cosmids or bacteriophages,comprising at least one DNA as defined above.

It also provides host cells transformed by at least one DNA as definedabove.

Other host cells of the invention comprise genes or gene fragmentsspecific to Nm, and are characterized in that their chromosome isdeleted by at least one DNA according to the invention, in particular aDNA responsible for the pathogenicity. They are more specificallybacterial cells, in particular of Nm.

The invention also relates to the RNAs of which the sequence correspondsin all or part to the transcription of at least one DNA sequence orsequence fragment as defined above.

The invention also relates to the antisense nucleic acids of the DNAs asdefined above, or of fragments of these DNAs.

These antisense nucleic acids carry, where appropriate, at least onesubstituent, such as a methyl group and/or a glycosyl group.

Other products which fall within the context of the invention includepolypeptides.

These polypeptides are characterized in that they have an amino acidchain corresponding to all or part of a sequence coded by the nucleicacids defined above, or deduced from sequences of these nucleic acids.

They are advantageously polypeptides corresponding to all or part of thepolypeptides exported beyond the cytoplasmic membrane, more specificallypolypeptides corresponding to all or part of those coded by a conservedregion.

As a variant, the polypeptides of the invention can be modified withrespect to those corresponding to the nucleic acid sequences such thatthey are particularly suitable for a given use, in particular use as avaccine.

Modification is understood as meaning any alteration, deletion orchemical substitution where this does not affect the biochemicalproperties of the corresponding natural polypeptides, more specificallyof functional proteins exported at the periplasm and the externalmembrane.

Other products according to the invention include antibodies directedagainst the above polypeptides.

The invention thus provides polyclonal antibodies, and also monoclonalantibodies, characterized in that they recognize at least one epitope ofa polypeptide as described above.

It also relates to fragments of these antibodies, more particularly thefragments Fv, Fab and Fab′2.

The invention also relates to the anti-antibodies which are capable ofrecognizing the antibodies defined above, or their fragments, by areaction of the antigen-antibody type.

According to the invention, the various products considered above areobtained by a synthesis and/or biological route in accordance withconventional techniques.

The nucleic acids can also be obtained from banks made up of Nm-specificDNAs such as are formulated by a subtractive technique, this techniquecomprising:

-   -   mixing of two DNA populations,    -   realization of at least one subtractive        hybridization-amplification iteration, and    -   collection of the desired DNA or DNAs, followed, where        appropriate, by its/their purification with elimination of        redundant sequences.

According to the invention, the two DNA populations originaterespectively from a strain of Neisseria meningitidis, the so-calledreference strain for which the specific bank must be constructed, and astrain of Neisseria, the so-called subtraction strain, having a homologyin primary DNA sequences of greater than about 70% with the Neisseriameningitidis strain, the DNA sequences of the subtraction and referencestrains being obtained respectively by random shearing, and by cleavageby a restriction endonuclease capable of producing fragments less thanabout 1 kb in size.

The invention provides in particular a process for obtaining Neisseriameningitidis-specific DNA banks, comprising the stages of

-   -   random shearing of the chromosomal DNA of a strain of Neisseria        gonorrhoeae, the so-called subtraction strain, in particular by        repeated passage through a syringe,    -   cleavage of the chromosomal DNA of a strain of Neisseria        meningitidis, the so-called reference strain, preferably by a        restriction enzyme producing fragments less than about 1 kb in        size,    -   splicing of the DNA fragments of the reference strain, cleaved        by the restriction enzyme, with suitable oligonucleotide        primers,    -   realization of a subtractive hybridization-amplification        iteration, by:        -   mixing of the two DNA populations under suitable conditions            for hybridization of homologous sequences, and then        -   amplification of auto-reannealed fragments and collection of            these fragments,        -   digestion of these fragments by a restriction enzyme and            re-splicing with oligonucleotide primers, followed by a    -   purification of the spliced DNA and, where appropriate, a new        iteration of the subtractive hybridization, comprising mixing of        DNA fragments of Neisseria gonorrhoeae sheared as indicated        above with DNA fragments of Neisseria meningitidis produced by        the preceding iteration, followed, if desired, by cloning of the        DNAs of the bank.

The primers used are oligodeoxynucleotide primers which are suitable forthe restriction endonuclease used and allow insertion into a cloningsite, such as the EcoRI site of the plasmid pBluescript. Such primerswill advantageously be chosen among the oligodeoxynucleotides referredto in the sequence listing under SEQ ID no. 36 to 45.

The banks thus obtained are formed from DNAs which are specific tomeningococci and absent from gonococci.

The specificity of the DNAs was verified, as described in the examples,at each iteration by Southern blots, with genes common to thesubtraction strain and to the reference strain, or with the total DNA ofeach of the strains digested by a restriction endonuclease, such asClaI.

At each iteration, the exhaustivity of the DNA bank was also verified bySouthern blotting with probes known to be specific to the referencestrain, that is to say for Neisseria meningitidis the frp, opc androtamase genes in particular.

The experiments carried out showed that the banks obtained by theprocess of the invention are deficient in genes having a significanthomology with species of Neisseria other than Neisseria meningitidis,for example the ppk or pilC1 genes, generally in only 2 or 3 iterations.

If necessary, two routes, which are not exclusive of each other, can betaken.

It is possible to proceed with an (n+1)^(th) iteration using the DNA ofiteration n as the DNA population of the reference strain.

As a variant, a second bank independent of the first is constructed,with a restriction enzyme of different specificity to that used in thefirst bank, for example MboI.

In all cases, it is preferable to keep each of the products produced byeach of the iterations performed.

The invention also provides the use of the subtractive techniquedescribed above to obtain banks of the DNAs common to Nm and Ng, butspecific with respect to Nl.

Three different banks are advantageously constructed, two of them bydigestion of the chromosomal DNA of Nm by MboI and Tsp5091, and thethird by digestion of the chromosomal DNA of Nm with MspI. Twosubtraction series allow the DNAs having the required specificity to becollected, as described in the examples.

The invention also relates to the process for obtaining these banks andthe banks themselves.

It can be seen that, generally, the process of the invention can be usedto obtain banks of DNAs specific to a given cell species, or to a givenvariant of the same species, where another species or another variantwhich is close genomically and expresses different pathogenic potenciesexists.

Using the process of the invention, DNA banks specific to given speciesof cryptococci, Haemophilus, pneumococci or also Escherichia coli, ormore generally any bacterial agent belonging to the same species andhaving different pathovars will advantageously be constructed.

Furthermore, from these banks the invention provides the means to haveavailable virulence factors specific to a species or a given variant.

Such banks are therefore tools which are of great interest for havingavailable attributes which are responsible for the specificity of apathogen, this use being more specifically illustrated according to theinvention by the obtaining of banks comprising the attributesresponsible for the specificity of the meningococcal pathogenesis.

Study of the products of the invention, the nucleic acids, polypeptidesand antibodies, has enabled an absolute specificity with respect otNeisseria meningitidis, regardless of the strain and its variability, tobe demonstrated.

These products are therefore particularly suitable for diagnosis orprevention of infections and meningitis caused by Neisseriameningitidis, whether in adults or children and regardless of theserogroups of the strain in question.

The method for diagnosis, according to the invention, of a meningococcalinfection, and more particularly of meningococcal meningitis, bydemonstration of the presence of Neisseria meningitis in an analyticalsample is characterized by the stages of:

-   -   bringing into contact a sample to be analysed, that is to say a        biological sample or a cell culture, and a reagent formulated        from at least one nucleic acid as defined above, if appropriate        in the form of a nucleotide probe or a primer, or, as a variant,        from at least one antibody or a fragment of an antibody as        defined above, under conditions which allow, respectively,        hybridization or a reaction of the antigen-antibody type, and    -   detection of any reaction product formed.

If the reagent is formulated from a nucleic acid, this can be in theform of a nucleotide probe in which the nucleic acid or a fragment ofthe latter is labelled in order to enable it to be detected. Suitablemarkers include radioactive, fluorescent, enzymatic or luminescentmarkers.

As a variant, the nucleic acid is included in a host cell, which is usedas the reagent.

In these various forms, the nucleic acid is used as such or in the formof a composition with inert vehicles.

If the reagent is compiled from an antibody, or a fragment of anantibody, this can be labelled for detection purposes. Most generally, afluorescent, enzymatic, radioactive or luminescent marker is used.

The antibody or the antibody fragment used, which is labelled ifappropriate, can be used as such or in the form of a composition withinert vehicles.

The sample used in the stage of bringing the components into contact isa biological sample produced by a mammal, such as cephalorachidianfluid, urine, blood or saliva.

The detection stage is carried out under conditions which allow thereaction product to be demonstrated when it is formed. Conventionalmeans use fluorescence, luminescence, colour or radioactive reactions,or also autoriadography [sic] techniques. It is also possible toquantify the product.

The invention also relates to the labelled products, the nucleic acidsand antibodies, as new products.

The method defined above can be used for diagnosis of an immune reactionspecific to a meningococcal infection.

The reagent used is thus a polypeptide according to the invention, ascoded by the said nucleic acid sequences, corresponding to the naturalproduct or a polypeptide which is modified but has the biological andimmunological activity of the corresponding natural polypeptide.

It is advantageously a polypeptide exported beyond the cytoplasmicmembrane of Neisseria meningitidis, more particularly the part of such apolypeptide corresponding to the conserved region of the DNA.

The invention also relates to kits for carrying out the methods definedabove. These kits are characterized in that they comprise:

-   -   at least one reagent as defined above, that is to say of the        nucleic acid, antibody or polypeptide type,    -   products, in particular markers or buffers, which enable the        intended nucleotide hybridization reaction or immunological        reaction to be carried out, as well as use instructions.

The specificity of the products of the invention and their location onthe chromosome of Neisseria meningitidis Z2491, either grouped in aregion and able to be interpreted as pathogenicity islets, or isolatedon the chromosome, impart to them a very particular interest forrealization of vaccine compositions with a universal purpose, that is tosay whatever the strain and the variability which it expresses. Thesecompositions can include in their spectrum other prophylaxes, and canbe, for example, combined with childhood vaccines.

The invention thus provides vaccine compositions which include in theirspectrum antimeningococcal prophylaxis, intended for prevention of anyinfection which may be caused by Neisseria meningitidis, thesecompositions being characterized in that they comprise, in combinationwith (a) physiologically acceptable vehicle(s), an effective amount ofpolypeptides or anti-antibodies or their fragments as defined above,these products optionally being conjugated, in order to reinforce theirimmogenicity [sic].

Immunogenic molecules which can be used comprise the poliovirus protein,the tetanus toxin, or also the protein produced by the hypervariableregion of a pilin.

As a variant, the vaccine compositions according to the invention arecharacterized in that they comprise, in combination with (a)physiologically acceptable vehicle(s), an effective amount:

-   -   of nucleic acids as defined above,    -   of transformed host cells as defined above, or    -   of Nm cells, the chromosome of which has been deleted        by at least one DNA sequence according to the invention involved        in the pathogenicity of the bacterium. The nucleotide material        used is advantageously placed under the control of a promoter of        its expression in vivo and synthesis of the corresponding        protein. To reinforce the immunogenicity, it is also possible to        combine this nucleic material with a DNA or an RNA which codes        for a carrier molecule, such as the poliovirus protein, tetanus        toxin or a protein produced by the hypervariable region of a        pilin.

The vaccine compositions of the inventions can be administeredparenterally, subcutaneously, intramuscularly or also in the form of aspray.

Other characteristics and advantages of the invention are given in theexamples which follow for illustration thereof, but without limiting itsscope.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

In these examples, reference will be made to FIGS. 1 to 11, which show,respectively,

FIGS. 1A, 1B, 1C, 1D, 1E, 1F and 1G: analysis of the subtractive bankTsp5091,

FIG. 2: the distribution of the Nm-specific sequences, in contrast toNg, on the chromosome of the strain Z2491 (left-hand part) and ofNm-specific sequences, in contrast to Nl (right-hand part),

FIGS. 3A to 3C: the reactivity of the clones of the 3 regions of thechromosome according to the invention towards a panel of strains of thegenus Neisseria,

FIG. 4: the position in region 2 of the chromosome of Nm ofoligonucleotides used as probes,

FIGS. 5, 6 and 7: the Southern blots of a panel of strains of the genusNeisseria, using parts of region 2 of Nm as probes,

FIGS. 8A to 8C: the Southern blots with 3 subtractive banks over a panelof 12 strains of Neisseria, and

FIGS. 9, 10 and 11: the reactivity of clones of the 3 subtractive bankswith respect to Nm, Nl and Ng.

In the examples which follow, the following materials and methods wereused:

-   Bacterial strains—To obtain the subtractive banks, strain Z2491 of    Nm (Achtman et al., 1991, J. Infect. Dis. 164, 375-382), the strains    MS11 (Swanson et al., 1974, Infect. Immun. 10, 633-644) and the    strains 8064 and 9764 of Nl were used, it being understood that any    other strain of the species in question could be used.

In order to verify the specificity of these banks, 6 strains of Nm, 4strains of Ng, one strain of Nl (Neisseria lactamica) and one strain ofNc (Neisseria cinerea) were used.

The six strains of Nm are: Nm Z2491 of serogroup A, Nm 8013 of serogroupC (XN collection), Nm 1121, no serogrouping possible (XN collection), Nm1912 serogroup A (XN collection), Nm 7972 of serogroup A (XN collection)and Nm 8216 of serogroup B (XN collection).

The four strains of Ng are: Ng MS11 (Pasteur Institute, Paris), Ng 403(Pasteur Institute, Paris), Ng 6934 (Pasteur Institute, Paris), Ng WI(isolated from a disseminated gonococcal infection), Ng 4Cl, Ng 6493 andNg FA 1090.

The strains of Nl are Nl 8064 and Nl 9764 (XN collection), and that ofNc is Nc 32165 (XN collection).

Molecular Genetics Techniques

Unless indicated otherwise, the techniques and reagents used correspondto those recommended by Sambrook et al (Sambrook et al 1989, MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press). Theoligodeoxynucleotides used in this study are:

(SEQ ID No.54) RBAm12, 3′ AGTGGCTCCTAG 54 (SEQ IN No.55) RBam24,5′ AGCACTCTCCAGCCTCTCACCGAG 3′; (SEQ ID No.60) Jbam12, 3′ GATCCGTTCATG5′; (SEQ ID No.61) JBAM24, 5′ ACCGACGTCGACTATCCATGAACG 3′; (SEQ IDNo.56) REco12, AGTGGCTCTTAA; (= RBam 24,SEQ ID NO.55) REco24,5′ AGCACTCTCCAGCCTCTCACCGAG 3′; (SEQ ID No.62) JEco12, GTACTTGCTTAA;(= JBam 24) JEco24, 5′ ACCGACGTCGACTATCCATGAACG 3′; (SEQ ID No.64)NEco12, AATTCTCCCTCG; (SEQ ID No.65). NEco24, AGGCAACTGTGCTATCCGAGGGAG;Transfer to Membranes (Southern Blots)

The transfers to membranes were effected by capillary transfers topositively charged nylon membranes (Boehringer Mannheim). Thehybridizations were carried out at 65° C. in a solution comprising NaPi[sic] 0.5 M pH 7.2/EDTA 1 mM/SDS 7%/BSA 1%. The membranes were washed ina solution comprising NaPi [sic] 40 mM pH 7.2/EDTA 1 mM/SDS 1%. Thefinal washing was carried out at 65° C. for 5 min.

The probe frp obtained with oligonucleotides based on the frpA sequencecorresponds to 2.4 kb of the 5′ end of the gene of the strain Z2491. Theopc and rotamase probes corresponding to whole genes are produced fromthe strain Z2491 using oligonucleotides constructed on the basis ofpublished sequences. The probes pilCl and ppk (polyphosphate kinase)correspond to inserts of the plasmids pJL1 and pBluePPK6001respectively.

EXAMPLE 1 Construction of Banks of DNAs Present in Nm and Absent from Ng

a. “MboI” Bank

Construction—The DNA of Nm Z2491 was cleaved by the endonuclease MboIand subjected to two iterations of a method called CDA (comprehensivedifference analysis) below. This method comprises subtractivehybridization in the presence of excess sheared DNA of Ng MS11 andamplification by PCR of those meningococcal sequences which, since theyare absent from or do not have significant homology with the DNA of NgMS11, could reanneal.

The chromosomal DNA of the strain Ng MS11 is sheared randomly byrepeated passage through a hypodermic syringe until fragments of a sizeranging from 3 to 10 kb are obtained. These DNA fragments are purifiedby extraction with phenol.

The chromosomal DNA of the strain Nm Z2491 is itself cleaved by therestriction endonuclease MboI. These DNA fragments (20 μg) are splicedwith 10 nmol of annealed oligonucleotides RBam12 and RBam24. The excessprimers are removed by electrophoresis over 2% agarose gel of lowmelting point. The part of the gel containing amplified fragmentsgreater than 200 bp in size is excised and digested by β-agarase. Thesefragments are purified by extraction with phenol.

To carry out a subtractive hybridization (first iteration), 0.2 μg ofthe Nm DNA spliced with the RBam oligonucleotides is mixed with 40 μg NgDNA in a total volume of 8 ml of a buffer EE 3× (a buffer EE 1× iscomposed of N-(2-hydroxyethyl)piperazine-N′-(3-propanesulphonic acid) 10mM and EDTA 1 mM, and its pH is 8.0). This solution is covered withmineral oil and the DNA is denatured by heating at 100° C. for 2 min. 2μl NaCl 5 M are added and the mixture is left to hybridize at 55° C. for48 h. The reaction mixture is diluted to 1/10 in a preheated solutioncomposed of NaCl and buffer EE, and in then immediately placed on ice.

10 μl of this dilution are added to 400 μl of PCR reaction mixture(Tris.HCl pH 9.0 10 mM; KCl 50 mM; MgCl₂ 1.5 mM; Triton X100 0.1%; 0.25mM of each of the four triphosphate deoxynucleotides; Taq polymerase 50units per ml). The mixture is incubated for 3 min at 70° C. to completethe ends of the reannealed meningococcal DNA fragments.

After denaturing at 94° C. for 5 min and addition of the oligonucleotideRBam24 in an amount of 0.1 nmol per 100 μl, the hybridizations areamplified by PCR (30 cycles of 1 min at 94° C., 1 min at 70° C. and 3min at 72° C., followed by 1 min at 94° C. and 10 min at 72° C.;Perkin-Elmer GeneAmp 9600).

The amplified meningococcal fragments are separated from the primers andhigh molecular weight gonococcal DNAs on gel. They are digested by MboIand the oligonucleotides JBam12 and JBam 24 are spliced to them again.These spliced DNAs are again purified over gel and extracted withphenol.

A second iteration of the subtractive hybridization is carried out on 40μg of the randomly sheared Ng DNA and 25 ng of the DNA spliced with theJBam oligonucleotides obtained from the first iteration of thesubtractive hybridization. During this second iteration, amplificationof the auto-annealed Nm DNA is effected with the aid of theoligonucleotide JBam24.

Specificity—In order to confirm their Nm specificity, the amplifiedsequences after the second iteration of the CDA method are labelled andused as a probe for the DNA digested by ClaI produced from a panel ofsix strains of Neisseria meningitidis, four of Neisseria gonorrhoeae,one of Neisseria lactamica and one of Neisseria cinerea.

The Southern blots obtained show that the amplified sequences resultingfrom the second iteration of the CDA method have a high reactivity withseveral bands corresponding to meningococci, and do not have areactivity with the bands corresponding to the Ng, Nl and Nc strains.

The “MboI” bank thus appears to be Nm-specific.

Exhaustivity—In order to test the exhaustivity of the bank, all theproducts produced from the first and second iterations of the CDA methodand also the initial chromosomal materials of Nm Z2481 [sic] and Ng MS11are subjected to agarose gel electrophoresis, transferred to a membraneand brought into contact with probes comprising genes known to bemeningococcus-specific, that is to say frp, opc and rotamase (Southernblotting).

As a result of these hybridizations, the Nm-specific gene frp isrepresented in the MboI bank by a fragment of 600 bp, but no activity isobserved for the rotamase and opc genes. The MboI bank, althoughNm-specific, therefore cannot be considered exhaustive.

Given their high specificity, the fragments produced by the seconditeration of the CDA method for the MboI bank can nevertheless be clonedon the BamHI site of the plasmid pBluescript.

A sequence corresponding to any of the Nm-specific genes can be includedin the subtractive bank only if it is carried by a restriction fragmentof appropriate size. This condition is a function of two factors.Firstly, the probability that the largest fragments are entirelyNm-specific is low. Secondly, even if such fragments existed, they wouldbe under-represented in the bank because of the limitations of the PCRtechnique, the amplification effectiveness of which decreases withincreasing size of the fragments. Fragments greater that about 600 bp insize are not included in the bank. Because of the absence of Mbofragments of suitable size from the chromosome of Nm Z2491, the rotamaseand opc genes cannot be included in the bank. Any enzyme cannot byitself produce a small fragment corresponding to any Nm-specific gene. Asecond bank was therefore constructed using another restriction enzymewith a different specificity: Tsp509 [sic].

b. “Tsp5091” Bank

Construction—The enzyme Tsp5091 has the advantage of producing fragmentsof smaller size (less than about 1 kb) than the enzyme MboI.

Tsp509I recognizes the sequence AATT and leaves, projecting at 5′, asequence of 4 bases compatible with EcoRI. The oligonucleotides used areReco, Jeco and NEco.

The method followed conforms with that followed for construction of the“MboI” bank described above. However, higher quantities of meningococcalDNA were used for the first iteration of the subtractive hybridizationin order to compensate for the higher number of fragments of lowmolecular weight produced by Tsp509I. For the first iteration, 400 ng NmDNA fragments and, in the second, 25 ng Nm fragments are subjected tosubtractive hybridization with 40 μg randomly sheared Ng DNA.

For the construction of this “Tsp509I” bank, as a control, a thirditeration of the subtractive hybridization is carried out using 40 μgsheared Ng DNA and 0.2 ng Nm fragments resulting from a digestion byTsp509I and a resplicing, with NEco adaptors, of the fragments obtainedas a result of the second iteration.

Specificity—As described for the previous bank, the product resultingfrom the second iteration of the CDA method is labelled and used as theprobe for a panel of strains of Neisseria.

FIG. 1A illustrates the Southern blot hybridization of products of thesecond iteration of the CDA method with the DNA digested by ClaI of: Nmin track a, Ng MS11 in track b, Nm 8013 in track c, Ng 403 in track d,Nm 1121 in track e, Ng 6934 in track f, Nm 1912 in track g, Ng WI(strain DGI) in track h, Nm 7972 in track i, Nl 8064 in track j, Nc32165 in track k, Nm 8216 in track 1.

In contrast to the high reactivity observed with all the Nm strains, alow or no reactivity is observed with the Ng, Nl and Nc strains.

The specificity of the bank was studied earlier by reacting membranetransfers (Southern blots) of the products produced by each of the threeiterations of the CDA method with probes corresponding to pilC1 and ppk.These two genes are common to Nm and Ng.

FIG. 1B shows an agarose gel after electrophoresis of the chromosomes ofNm Z2491 and Ng Ms11, digested by Tsp509 [sic], and products resultingfrom each of the iterations of the CDA method.

In track a 1 μg of the chromosome of Nm was deposited, in track b 1 μgof that of Ng, in track c 0.15 μg of the products resulting from thefirst CDA iteration, in track d 0.1 μg of those of the second iteration,in track e 0.05 μg of the third iteration, MW representing the molecularsize markers.

FIGS. 1C and 1D show gels obtained as described in FIG. 1B aftertransfer to the membrane (Southern blots) and hybridization with pilC1(FIG. 1C) and ppk (FIG. 1D).

At the end of the second iteration of the CDA method, the sequencescorresponding to the pilC1 and ppk genes are completely excluded fromthe bank.

Exhaustivity—The exhaustivity of the bank was examined by reacting theproducts resulting from the subtractive hybridization with the probescorresponding to three Nm-specific genes (frp, rotamase and opc).

These Nm-specific probes react with the amplification products resultingfrom the first and second iteration of the subtractive hybridization.

FIGS. 1E, 1F and 1G show gels obtained as described in FIG. 1B aftertransfer to the membrane (Southern blots) and hybridization with frpA(FIG. 1E), rotamase (FIG. 1F) and opc (FIG. 1G).

However, a third iteration of the subtractive hybridization leads to theloss of Nm-specific sequences, since the fragments which react with therotamase and opc genes are absent from this third iteration.

In consideration of all these data, it emerges that the productsresulting from the second iteration of the CDA method are Nm-specificand also constitute an exhaustive bank of Nm-specific sequences.

The products resulting from this second iteration are cloned at theEcoRI site of the plasmid pBluescript.

The bank produced by Tsp509I is more exhautive [sic] than the bankproduced by MboI, as the theory considerations based on the enzymaticproduction of smaller restriction fragments would suggest.

In accordance with this aspect, it should be noted that the Tsp509I bankis less redundant than the MboI bank, that is to say it comprises lessduplication of clones. 86% of the clones of the Tsp509I bank correspondto distinct sequences, while only 43% of the clones correspond todistinct sequences in the MboI bank (data not shown).

The bank produced by Tsp509I thus constitutes a source of Nm-specificclones.

EXAMPLE 2 Analysis of the Clones of the Subtractive Bank

Cloning and Sequencing of the Nm-Specific DNAs

The DNAs of the subtractive banks are clones at the BamHI (MboI bank) orEcoRI (Tsp509I bank) site of the plasmid pBluescript, and thentransformed in DH5α of E. coli. The inserts are amplified by PCR carriedout on the transformed colonies using the primers M13-50 and M13-40, thelatter primer being biotinylated on its 5′ end.

Sequencing was carried out on each PCR product after separation of thebiotinylated and non-biotinylated strands using the system of DynabeadsM-280 with streptavidin (Dynal, Oslo). The sequences are screenedaccording to their homologies with previously published sequences usingthe computer programs Blastn and Blastx (NCBI, USA and Fasta).

The PCR products resulting from the transformed bacteria colonies afterusing the primers M13-40 and M13-50 as described above were labelled byincorporation with random priming of α-³²P-dCTP and were used as a probefor the membrane transfers of the chromosomal DNA digested by ClaI ofstrains Nm Z2491 and Ng MS11, as described above, in order to verifytheir specificity.

Mapping of Clones on the Chromosome of the Strain Nm Z2491.

The results of studies carried out with 17 clones of the “MboI” bank(designated by the letter B) and 16 clones of the “Tsp5091” bank(designated by the letter E), each of these clones having a uniquesequence and being without counterpart in Ng, are reported.

The positions of the DNA sequences corresponding to cloned Nm-specificproducts were determined with respect to the published map of thechromosome of Nm Z2491 (Dempsey et al. 1995, J. Bacteriol. 177,6390-6400) and with the aid of transfers to membranes (Southern blots)of agarose gel subjected to pulsed field electrophoresis (PFGE).

The Nm-specific clones are used as probes for a hybridization onmembranes (Southern blots) of the DNA of Nm Z2491 digested with enzymesof rare cutting sites, that is to say PacI, PmeI, SgfI, BglII, SpeI NheIand SgfI.

The gels (20×20 cm) were gels of 1% agarose in a buffer TBE 0.5× andwere subjected to electrophoresis at 6 V/cm for 36 hours according topulsation periods varying linearly between 5 and 35 seconds.

The hybridizations on the membrane (Southern blots) were carried out asdescribed above.

The results obtained are shown on FIG. 2: the reactivity was located bycomparison with the positions of the fragments of corresponding size onthe published map. The positions of all the genetic markers mapped byDempsey et al (mentioned above) are visualized with the aid of points onthe to linear chromosomal map. The Nm-specific genes disclosedpreviously are labelled with an asterisk. The two loci called “frp”correspond to the frpA and frpc genes. The “pilC” loci correspond to thepilC1 and pilC2 genes, which are pairs of homologous genes and are notdistinguished on the map. The accuracy of the positions of theNm-specific clones of the invention depends on the overlapping ofreactive restriction fragments. On average, the position is +/−20 kb.

This mapping reveals a non-random distribution of the Nm-specificsequences. The majority of the Nm-specific sequences belong to threedistinct groups. One of these groups (region 1) corresponds to theposition of genes relating to the capsule which have been describedpreviously.

A distinction is made between:

-   -   E109, E138, B230 and B323 as being region 1,    -   B322, B220, B108, B132, B233, B328, E139, E145 as B101 as being        region 2, and    -   B306, E114, E115, E124, E146, E120, E107, E137 and 142 as being        region 3.        63% of the sequences identified as specific to meningococci are        located inside these three distinct regions.

This grouping contrasts with the distribution of previously disclosedNm-specific genes (frpA, frpC, porA, opc and the region relating to thecapsule).

This prior art would suggest in fact that the Nm-specific genes, withthe exception of functional genes relating to the capsule, weredispersed along the chromosome.

Mapping of Nm-specific sequences on the chromosome leads to anunexpected result with regard to the prior art.

The majority of the genetic differences between the meningococcal andgonococcal strains tested are grouped in three distinct regions.

Meningococcal genes relating to the capsule are grouped in region 1.

The function of genes of the other regions is unknown, but homologieswith published sequences (table 1) suggest similarities between certaingenes of region 3 and bacteriophage transposase and regulatory proteins.No meningococcal virus has been characterized and it is tempting tothink that these sequences are of phagic origin. Interestingly, thegenome of H. influenzae also contains a sequence homologous to that ofthe Ner regulatory protein of phage Mu, but it is not known if it is afunctional gene.

The clone B208 has a high homology (48% identical, 91% homology for 33amino acids) with a clone of conserved regions field III) in the classof proteins which bind to TonB-dependent ferric siderophors.

The proximity of this clone with the Nm-specific porA genes and the frpgenes regulated by iron, and in particular the possibility that it is anNm-specific receptor protein exposed on the external membrane in itselfis a good candidate for further research.

The clone B339 corresponds to the Nm-specific insertion sequence IS1106.

The low homology between the clone B134 and the Aeromonas insertionsequence and also the presence of multiple copies of the clone B134among the various strains of Nm suggest that it could be a new type ofNm-specific insertion sequence.

The possibility that the regions containing the Nm-specific clones couldcorrespond to pathogenicity islets as described previously for E. coliand Y. pestis is of particular interest.

The clones isolated in this invention will allow better understanding ofthe relevance of Nm-specific regions in allowing cloning and sequencingof larger chromosomal fragments, and directly by their use for locimutations.

Finally, detection of meningococcus-specific genes possibly involved inthe pathogenicity of the organism allows targeting of suitable antigenswhich can be used in an antimeningococcal vaccine.

The effectiveness and the speed of the method according to theinventions enables it to be used in a large number of situations forwhich the genetic differences responsible for a phenotype peculiar toone of 2 close pathogens are investigated.

Study of the Reactivity of the Clones of Regions 1, 2 and 3 Towards aPanel of Strains of Neisseria.

The PCR products corresponding to inserts of each of the clones werecollected and used as probes for hybridization on membranes (Southernblots) for a panel of strains of Nm, Ng, Nl and Nc.

Regions 1 and 2 produce a limited number of bands for each of themeningococci. This suggests that these regions are both Nm-specific andcommon to all the meningococci.

FIG. 3 illustrates the reactivity of the clones of regions 1, 2 and 3towards a panel of neisserial strains. The clones of regions 1 (FIG.3A), 2 (FIG. 3B) and 3 (FIG. 3C) taken together were used as probestowards a panel of meningococci, gonococci and towards a strain of Nland Nc.

The tracks are as follows: DNA of: Nm Z2491 in track a, of Ng MS11 intrack b, of Nm 8013 in track c, of Ng 403 in track d, of Nm 1121 intrack e, of Ng 6934 in track f, of Nm 1912 in track g, of Ng WI (strainDGI) in track h, of Nm 7972 in track i, of Nl 8064 in track j, of Nc32165 in track k, and of Nm 8216 in track 1.

Remarkably, region 3 has reactivity only with the meningococci ofserogroup A. This region 3 is therefore specific to a sub-group of Nm.

A comparison was made with the known sequences in the databanks in orderto evaluate the possible functions of the cloned regions.

Table 1 which follows gives the positions of specific clones on thechromosomal map and the homologies with known sequences.

TABLE 1 Position of specific clones on the chromosomal map andhomologies with known sequences Size Reactive Position Name of offragments on Homologies of protein clone* insert Pac Pmc Bg1 Spe Nhe SgfZ2491 sequences B305 259 18-20 15-17 22-23 18  11- 2 λ736 13 B333 23515-17 22-23 18  11- 2 λ736 13 E109¹⁺ 211 6-7 11-15 10  11- 2 tufAprotein LipB 13 ctrA N. meningitidis (3 × 10⁻²⁶) E138¹⁺ 315  1 6-7 11-1510  11- 2 tufA protein LipB 13 ctrA N. meningitidis (4 × 10⁻⁷⁵) B230¹356 1-3 6-7 1 10  11- 2 ctrA protein KpsC E. coli 13 (3 × 10⁻⁵³) B323¹363  1 6-7 1 10  11- 2 ctrA protein CtrB 13 N. meningitidis (2 × 10⁶⁴)B322² 210 2 16-18 6 1 5 pilQ/λ HlyB S. marcescens 740 (4 × 10⁻¹⁵) B220²341 2 16-18 6 ≧18 5 pilQ/λ 740 B108² 275 2 19-21 6 ≧18 5 pilQ/λ 740B132² 411  2 2 19-21 6 ≧18 5 pilQ/λ 740 B233² 164 1-3 2 19-21 6 ≧18 5pilQ/λ 740 B328² 256 1-3 2 22-23 6 ≧18 5 pilQ/λ 740 E139² 324  2 2 19-216 ≧18 5 pilQ/λ 740 E145² 343  2 2 19-21 6 ≧18 5 pilQ/λ 740 B101² 254≧20   2 19-21 6 ≧18 5 pilQ/λ 740 E103q 334 2 11-15 3-5 10 3 λ644B326^(§) 314 2 11-15 3-4 10 3 λ644 B326 (low 5 6 16  2 1 argFreactivity) B342 167 2 19  3-4 6-7 3 iga E136 249 2 7 1 3 3 lepA B208177 1 2 3-4 2 1 porA FeIII pyochelin receptor P. aeruginosa (5.10⁻⁴) =B306^(3#) 219 11 5 11-12 5 2 4 parC E114³ 227 11 5 11-12 5 2 4 parCE115^(3#) 251 5 11-15 5 2 4 parC E124³ 208 5 11-12 5 2 4 parC E146³ 1465 11-15 5 4 parC E120³ 263 5 3-4 5 16 4 opaB E107³ 248 11 14-17 3-4 5 164 opaB E137³ 274 14-17 3-4 5 16 4 opaB Transposase Bacteriophage D3112(6 × 10⁻¹²) E142³ 230 14-17 3-4 5 16 4 opaB Protein Ner-Likc H.influenzae (6 × 10⁻²³) Protein binding to the DNA Ner, phage mu (3 ×10⁻¹⁸) E116 379 5-7 11-13 3-4 2 6-7 8 λ375 B313 436  9 9 3-4 13- 5 2λ611 14 B341 201 8-10 9 3-4 13- 5 2 λ611 14 E102 238 11-13 3-4 19  5 2λ601 Hypothetical protein H11730 H. influenzae (7 × 10⁻²⁴) B134 428multiple transposase ISAS2 Aeromonas salmonicida (5 × 10⁻⁵) B339 259multiple transposase IS 1106 N. meningitidis (6 × 10⁻⁴⁵) The level ofhomologies found, as given by the Blastx program, are indicated inparentheses *The clones labelled with the index “1”, “2” or “3” belongto regions “1”, “2” or “3” respectively of the chromosome of N.meningitidis Z2491. ¹⁺E109 and E138 are contiguous clones ^(§)B306 andE115 overlap ^(#)B236 also has a low reactivity in the region of arg Fq) Clone E103 contains a Tsp509 I site and can therefore contain twoinserts; however, since it reacts only with a single fragment ClaI (Oks)of the chromosome of N. meningitidis Z2491 and occupies only oneposition on the map, this clone is included here.

Firstly, it can be seen that the clones of region 1 all correspond togenes involved in biosynthesis of the capsule. These genes havepreviously been studied among the Nm of serogroup B (Frosch et al. 1989,Proc. Natl. Acad. Sci. USA 86, 1669-1673 and Frosch and Muller 1993,Mol. Microbiol. 8 483-493).

With the exception of a low homology with the haemolysin activator ofSerratia marcescens, the clones of region 2 have no significant homologywith published sequences, either in the DNA or the proteins.

Two of the clones of region 3 have interesting homologies with proteinswhich bind to the DNA, in particular the bacteriophage regulatoryproteins and transposase proteins.

Clone B208 has a high homology with one of the conserved regions in oneclass of receptors (TonB-dependent ferric siderophor).

Clones B134 and B339 hybridize with several regions of the chromosome(at least 5 and at least 8 respectively).

Data relating to the sequences show that clone B339 corresponds to theNm-specific insertion sequence S1106.

The translation of the clone B143 has a limited homology with thetransposase of an Aeromonas insertion sequence (SAS2) (Gustafson et al.1994, J. Mol. Biol. 237, 452-463). We were able to demonstrate bytransfer on a membrane (Southern blots) that this clone is anNm-specific entity present in multiple copies in the chromosomes ofevery meningococcus of the panel tested.

The other clones have no significant homology with the publishedneisserial sequences, and furthermore nor with any published sequence.These clones therefore constitute, with the majority of the other clonesisolated, a bank of totally new Nm-specific loci.

EXAMPLE 3 Study of Region 2 of the Nm Chromosome

Determination and Characterization of the Sequence of Region 2

PCR amplification is carried out with the chromosomal DNA of strainZ2491 of serogroup A, sub-group IV-1 using oligonucleotide primersformulated from each of the sequences of clones of region 2 in severaldifferent combinations. The PCR products which overlap are sequencedfrom the 2 strands using the chain termination technique and automaticsequencing (ABI 373 or 377).

To prolong the sequence beyond the limits of the clones available,partial SauIIIA fragments of 15 kb of the strain Z2491 are cloned inLambda DASH-II (Stratagene).

The phages containing the inserts overlapping region 2 are identified byhybridization with clones of this region as probes. The DNA inserted issequenced from the ends of the inserts, and these sequences are used toformulate new primers which will serve to amplify the chromosomal DNAdirectly, and not the phagic DNA.

An amplification of the chromosomal DNA is obtained using these newprimers and those of the sequence previously available.

These PCR products are also sequenced from the 2 strands, which leads toa complete sequence of 15,620 bp (SEQ ID No. 36). The reading frames ofthis sequence which start with ATG or GTG and are characterized by ahigh codon usage index are analysed.

This analysis reveals 7 ORFs of this type which fill the major part ofthe sequence of 15,620 bp. The positions of these ORFs are thefollowing:

ORF-1: 1330 to 2970 (SEQ ID No. 37); ORF-2: 3083 to 9025 (SEQ ID No.38); ORF-3: 9044 to 9472 (SEQ ID No. 39); ORF-4: 9620 to 12118 (SEQ IDNo. 40); ORF-5: 12118 to 12603 (SEQ ID No. 42); ORF-6: 12794 to 13063(SEQ ID No. 43); ORF-7: 13297 to 14235 (SEQ ID No. 44); and ORF-8: 14241to 15173 (SEQ ID No.45).

ORF-4 starts with the codon GTG and overlaps a slightly smaller ORF (SEQID No. 41) in the same reading frame (10127-12118, frame 2), whichstarts with the codon ATG.

ORF-4 codes for a protein which has structural homologies with a familyof polypeptides comprising pyocins (Pseudomonas aeruginosa), collcinsand intimins (Escherichia coli), which are bactericidal toxins (pyocins,collcins) or surface proteins involved in adhesion of bacteria toeukaryotic proteins. ORF-7 encodes a protein, the sequence of whichcontains a potentially transmembrane region and which has structuralhomologies with periplasmic proteins or proteins inserted in theexternal membrane of bacteria. The structural homologies of ORF-4 andORF-7 have been identified with the aid of the PropSearch program.

Investigation of sequences homologous to other ORFs in GenBank with theaid of the BLAST program revealed a homology between the N-terminalregions of ORF-2 and filamentous haemagglutinin B of Bordetellapertussis (43% similarity, 36% identical over 352 amino acids) andbetween ORF-1 and the protein fhaC of Bordetella pertussis (35%similarity, 27% identical over 401 amino acids). ORF-1 and ORF-2 areneighbouring genes in the strain Z249I and filamentous haemagglutinin Bof Bordetella pertussis and fhaC are neighbouring genes in Bordetellapertussis, which reinforces the probability that these homologiesreflect functional homologies.

Confirmation of the specificity of region 2 with respect to Nm

Southern blots are carried out using the DNA probes obtained by PCRamplification of various parts of region 2 using oligonucleotide primersformulated from sequences of clones of region 2.

The approximate position of these oligonucleotides is shown on FIG. 4.

These are the oligonucleotides called R2001 (SEQ ID No. 46) and R2002(SEQ ID No. 47) in one half of ORF-1, the oligonucleotides b332a (SEQ IDNo. 48), e139a (SEQ ID No. 49), b132a (SEQ ID No. 50) and b233b (SEQ IDNo. 51) in one half of ORF-1+the majority of ORF-2, and theoligonucleotides e145a (SEQ ID No. 52) and b101a (SEQ ID No. 53) in ⅓ ofORF-4+ORF-5 to 7.

The three Southerns are carried out under the following hybridizationconditions:

-   16 h at 65° C.,-   NaPO₄ 0.5 M, pH 7.2-   EDTA-Na 0.001 M-   1% sodium dodecylsulphate.

For the washing, heating is carried out for 10 min at 65° C., and NaPO₄0.5 M, pH 7.2; EDTA-Na 0.001 M, 1% sodium dodecylsulphate are used.

FIGS. 5, 6 and 7 respectively show the Southern blots obtained with eachof the abovementioned ORF parts.

The 14 tracks correspond respectively, in each of the Southerns, to

-   1: MS11 (Ng)-   2: 403 (Ng)-   3: FA1090 (Ng)-   4: W1 (Ng)-   5: 6493 (Ng)-   6: marker (lambda hindIII)-   7: Z2491 (Nm, gpA)-   8: 7972 (Nm gpA)-   9: 8013 (Nm, gpC)-   10: 1121 (Nm, grouping not possible)-   11: 1912 (Nm, gpB)-   13: 32165 (Nc)-   14: 8064 (Nl).

Given that a panel of strains of Neisseria is used in these experimentsand that each well is charged with a similar amount of digested DNA,these 3 Southern blots clearly show that the sequences corresponding toregion 2 are found in all the meningococci tested and that significanthomologous sequences do not exist in the genome of the Ng of the strainstested.

EXAMPLE 4 Identification of Regions of the Nm Genome Absent from Nl andCommon with Ng

The technique described in example 1 is followed, but the chromosomalDNA of one strain of Nm (Z2491) and 2 strains of Nl (XN collections),equal parts of the DNAs of which are mixed, is used.

2 subtractions are performed using the R and J series of primers. Threedifferent banks are thus obtained.

Two banks, called Bam and Eco, are obtained respectively by digestion ofthe chromosomal DNA of Nm Z2491 by MboI and Tsp5091; a third bank,called Cla, which results from digestion of the chromosomal DNA of Nm byMspI, is obtained using the primer set RMsp10, RMsp24, JMsp10 andJMsp24. All the primers used are shown in the following table 2.

TABLE 2 Adapters for differential banks Chromosomal DNA Cloning indigested by pBluescript by MboI → BamHI Tsp509I → EcoRI MspI → ClaI

First subtraction cycle (SEQ ID No.54) RBam12: 3′ AGTGGCTCCTAG 5′ (SEQID No. 55) RBam24: 5′ AGCACTCTCCAGCCTCTCACCGAG 3′ (SEQ ID No.56) REco12:AGTGGCTCTTAA (SEQ ID No. 55) RBam24: 5′ AGCACTCTCCAGCCTCTCACCGAG 3′(REco 24 = RBam 24) (SEQ ID No.57) RMsp10: AGTGGCTGGC (SEQ ID No. 58)RMsp24: 5′ AGCACTCTCCAGCCTCTCACCGAC 3′ Second subtraction cycle (SEQ IDNo.59) Jbam12: 3′ GTACTTGCCTAG 5′ (SEQ ID No. 60) JBam24:5′ ACCGACGTCGACTATCCATGAACG 3′ (SEQ ID No. 61) JEco12: GTACTTGCTTAA (SEQID No 60) JBam24: 5′ ACCGACGTCGACTATCCATGAACG 3′ (JEco 24 = TBam 24)(SEQ ID No. 62) JMsp10: GTACTTGGGC (SEQ ID No. 63) JMsp24:5′ ACCGACGTCGACTATCCATGAACC 3′

After 2 subtractions, the entire product of each amplification islabelled and used as a probe.

The subtractive banks are checked by Southern blotting over a panel of12 strains of Neisseria (chromosomal DNA cut by ClaI). The hybridizationconditions are identical to those given in example 1.

These Southern blots are shown on FIGS. 8A to 8C, which relaterespectively to the MboI/BamHI bank, to the MspI/ClaI bank and to theTsp5091/EcoRI bank.

The 12 tracks correspond respectively, to

-   1: Nm Z2491 (group A)-   2: Nl 8064-   3: Nm 8216 (group B)-   4: Nl 9764-   5: Nm 8013 (group C)-   6: Ng MS11-   7: Nm 1912 (group A)-   8: Ng 4C1-   9: Nm 1121 (grouping not possible)-   10: Ng FAlO9O-   11: Nc 32165-   12: Nm 7972 (group A)

Examination of the Southern blots shows that the sequences contained ineach bank are specific to Nm and are not found in Nl. Furthermore, thereactivity found with the strains of Ng suggests that some of thesesequences are present in Ng.

Each of these banks was then cloned in pBluescript at the BamHI site forBam, or the EcoRI sit for Eco, or the ClaI site for Cla. In order toconfirm the specificity of the clones with respect to the Nm genome,restriction of the individual clones and radiolabelling thereof werecarried out. The clones showing reactivity for both Nm and Ng were keptfor subsequent studies. These clones are shown on FIGS. 9, 10 and 11,which give the profiles with respect to Nm, Nl and Ng of 5 clones of theBam bank (FIG. 9), 16 clones of the Eco bank (FIG. 10) and 13 clones ofthe Cla bank (FIG. 11).

These clones were sequenced using universal and reverse primers. Theyare

-   Bam clones    partial B11 of 140 bp (SEQ ID No. 66), partial B13 estimated at 425    bp (SEQ ID No. 67), B26 of 181 bp (SEQ ID No. 68), B33 of 307 bp    (SEQ ID No. 69), B40 of 243 bp (SEQ ID No. 70),-   Cla clones    C16 of 280 bp (SEQ ID No. 72), partial C20 estimated at 365 bp (SEQ    ID No. 73), partial C24 estimated at 645 bp (SEQ ID No. 74), partial    C29 estimated at 245 bp (SEQ ID No. 75), C34 of 381 bp (SEQ ID No.    76), C40 of 269 bp (SEQ ID No. 77), C42 of 203 bp (SEQ ID No. 78), p    C43 of 229 bp (SEQ ID No. 79), C45 of 206 bp (SEQ ID No. 80), C47 of    224 bp (SEQ ID No. 81), C62 of 212 bp (SEQ ID No. 82), and C130 (5′    . . . ) estimated at 900 bp (SEQ ID No. 83), and-   Eco clones    E2 of 308 bp (SEQ ID No. 84), partial E5 estimated at 170 bp (SEQ ID    No. 85), partial E22 estimated at 300 bp (SEQ ID No. 86), E23 of 273    bp (SEQ ID No. 87), E24 of 271 bp (SEQ ID No. 88), E29 of 268 bp    (SEQ ID No. 89), partial E33 estimated at 275 bp (SEQ ID No. 90),    partial E34 estimated at 365 bp (SEQ ID No. 91), E45 of 260 bp (SEQ    ID No. 92), E59 estimated at greater than 380 bp (SEQ ID No. 93),    E78 of 308 bp (SEQ ID No. 94), E85 of 286 bp (SEQ ID No. 95), E87 of    238 bp (SEQ ID No. 96), partial E94 greater than 320 bp (SEQ ID No.    97), partial E103 greater than 320 bp (SEQ ID No. 98) and E110 of    217 bp (SEQ ID No. 99).

Mapping of each clone was carried out on the chromosome of Nm Z2491 asdescribed in example 1. The results obtained are given on the right-handpart of FIG. 2. It is found that these clones correspond to regionscalled 4 and 5. These regions are therefore made up of sequences presentboth in Nm and in Ng, but not found in Nl. It is therefore regarded thatthese are sequences which code for virulence factors responsible for theinitial colonization and penetration of the mucosa. Region 4 is locatedbetween argF and regF on the chromosome of Nm 2491 [sic], and region 5is located between the lambda 375 marker and penA. This region probablycontains sequences which code for an Opa variant and a protein whichbinds transferrin.

A comparison with the known sequences in the databanks has half [sic]that in region 4 only the clone C130 has a homology, that is to say withMspI methylase. In region 5, no homology with known sequences was foundwith the clones C8, E2, B40, C45, E23 and E103. For the other clones,the homologies are the following:

B11 arginine decarboxylase SpeA; C29 arginine decarboxylase SpeA; C62oxoglutarate/malate transporter; repetitive DNA element; E34 repetitiveDNA element; E94 murine endopeptidase MepA; C47 citrate synthase PrpC;E78 citrate synthase PrpC

EXAMPLE 5 Demonstration of the Presence of One or More Strains ofNeisseria meningitidis in a Biological Sample

A biological sample of the cephalorachidian fluid, urine, blood orsaliva type is taken.

After filtration and extraction, the DNAs present in this sample aresubjected to gel electrophoresis and transferred to a membrane bySouthern blotting.

A nucleotide probe constructed by labelling SEQ ID No. 5 with ³²P isincubated with this transfer membrane.

After autoradiography, the presence of reactive band(s) allows diagnosisof the presence of Neisseria meningitidis in the sample.

EXAMPLE 6 Vaccine Composition Including in its SpectrumAntimeningocococcal Prophylaxis and Intended for Prevention of any formof Infection by Neisseria meningitidis

The peptide coded by a sequence including SEQ ID No. 10 is conjugatedwith a toxin.

This conjugated peptide is then added to a composition comprising theanti-Haemophilus and antipneumococcal vaccine, or any other childhoodvaccine.

After having been sterilized, the resulting composition can be injectedparenterally, subcutaneously or intramuscularly.

This same composition can also be sprayed on to mucosa with the aid of aspray.

1. An isolated DNA which is specific to Neisseria meningitidis (Nm) andNeisseria gonorrheae (Ng), and hybridizes on a Southern blot to SEQ IDNO:95 and does not hybridize on a Southern blot to a DNA sequence ofNeisseria lactamica (Nl) strain Nl8064, under the followinghybridization conditions: 18 h at 65° C., with a solution comprising 0.5M NaPO₄ pH 7.2, 0.001 M EDTA-Na, 1% bovine serum albumin and 7% sodiumdodocylsulphate, followed by at least two washes in a solutioncomprising 40 mM Na PO₄ pH 7.2, 1 mM EDTA, and 1% SDST, the final washbeing conducted at 85° C. for 5 minutes, or the complement of saidisolated DNA which is specific to Neisseria meningitidis (Nm) andNeisseria gonorrheae (Ng), provided that said DNA or the complement ofsaid isolated DNA is not pilC, or a gene involved in the biosynthesis ofany one of the polysaccharide capsule, IgA proteases, pilin, a proteinwhich binds transferin, a protein which binds lactoferin, and an opacityprotein said DNA being within an islet involved in the polonization ofthe nasopharynx or invasion of the submucousal space or systemicdissemination of Nm.
 2. A composition comprising the DNA or complementof claim 1 and a carrier.
 3. An isolated DNA which is specific toNeisseria memingitidis (Nm) and Neisseria gonorrheae (Ng), andhybridizes on a Southern blot to SEQ ID NO:95 and does not hybridize ona Southern blot to a DNA sequence of Neisseria lactamica (Nl) strainNl8064, under the following hybridization conditions: 16 h at 65° C.,with a solution comprising 0.5 M NaPO₄, pH 7.2, M EDTA-Na, 1% bovineserum albumin and 7% sodium dodecylsulphate, followed by at least twowashes in a solution comprising 40 mM Na PO₄ pH7.2, 1 mM EDTA and 1%SDS, the final wash being conducted at 65° C. for 5 minutes, or thecomplement of said isolated DNA which is specific to Neisseriameningitidis (Nm) and Neisseria gonorrheae (Ng), provided that said DNAor the complement of said isolated DNA is not pilC, or a gene involvedin the biosynthesis of any one of the polysaccharide capsule, IgAproteases, pilin, a protein which binds transferrin, a protein whichbinds lactoferin, and an opacity protein.
 4. A composition comprisingthe DNA or complement of claim 3 and a carrier.
 5. A method of detectingNeisseria meningitidis (Nm) or Neisseria gonorrheae (Ng) in a samplefrom a patient said method comprising providing isolated sample DNA ofsaid sample from the patient, contacting said sample DNA with theisolated DNA of claim 1, said contacting being performed underconditions which allow hybridization of said sample DNA and saidisolated DNA, and detecting any hybridization of said sample DNA andsaid isolated DNA, such that said hybridization of said sample DNA andsaid isolated DNA specifically indicates the presence of said Nm or Ngin said sample, wherein said isolated DNA is specific to Nm and Ng anddoes not hybridize on a Southern blot to a DNA sequence of Neisserialactamica (Nl) strain Nl8064 under the following hybridizationconditions: 18 h at 65° C., with a solution comprising 0.5 M NaPO₄ pH7.2, 0.001 M EDTA-Na, 1% bovine serum albumin and 7% sodiumdodecylsulphate, followed by at least two washes in a solutioncomprising 40 mM Na PO₄ pH 7.2, 1 mM EDTA, and 1% SDS, the final washbeing conducted at 65° C. for 5 minutes, and wherein said isolated DNAspecifically hybridizes to Nm DNA and Ng DNA in the presence of Nl DNA.6. A method of detecting Neisseria meningitidis (Nm) or Neisseriagonorrheae (Ng) in a sample from a patient, said method comprisingproviding isolated sample DNA of said sample from the patient,contacting said sample DNA with the composition of claim 1 underconditions which a low hybridization of said sample DNA and DNA in saidcomposition, and detecting any hybridization of said sample DNA and saidDNA in said composition, such that said hybridization of said sample DNAand said DNA in said composition specifically indicates the presence ofsaid Nm or Ng in said sample, wherein said DNA in said composition isspecific to Nm and Ng and does not hybridize on a Southern blot to a DNAsequence of Neisseria lactamica (Nl) strain Nl8064 under the followinghybridization conditions: 16 h at 65° C., with a solution comprising 0.5M NaPO₄ pH 7.2, 0.001 M EDTA-Na, 1% bovine serum albumin and 7% sodiumdodecylsulphate, followed by at least two washes in a solutioncomprising 40 mM Na PO₄ pH 7.2, 1 mM EDTA, and 1% SDS, the final washbeing conducted at 65° C. for 5 minutes, and wherein said DNA is saidcomposition specifically hybridizes to Nm DNA and Ng DNA in the presenceof Nl DNA.
 7. A method of detecting Neisseria meningitidis (Nm) ofNeisseria gonorrheae (Ng) in a sample from a patient, said methodcomprising providing isolated sample DNA of said sample from thepatient, contacting said sample DNA with the isolated DNA of claim 3,said contacting being performed under conditions which allowhybridization of said sample DNA and said isolated DNA, and detectingany hybridization of said sample DNA and said isolated DNA, such thatsaid hybridization of said sample DNA and said isolated DNA specificallyindicates the presence of said Nm or Ng in said sample, wherein saidisolated DNA is specific to Mm and Ng and does not hybridize on aSouthern blot to a DNA sequence of Neisseria lactamica (Nl) strainNl8064 under the following hybridization conditions: 16 h at 65° C.,with a solution comprising 0.5 M NaPO₄ pH 7.2, 0.001 M EDTA-Na, 1%bovine serum albumin and 7% sodium dodecylsulphate, followed by at leasttwo washes in a solution comprising 40 mM Na PO₄pH 7.2, 1 mM EDTA, and1% SDS, the final wash being conducted at 65° C. for 5 minutes, andwherein said isolated DNA specifically hybridizes to Nm DNA and Ng DNAin the presence of Nl DNA.
 8. A method of detecting Neisseriameningitidis (Nm) or Neisseria gonorrheae (Ng) in a sample from apatient, said method comprising providing isolated sample DNA of saidsample from the patient, contacting said sample DNA with the compositionof claim 4 under conditions which allow hybridization of said sample DNAand DNA in said composition, and detecting any hybridization of saidsample DNA and said DNA in said composition, such that saidhybridization of said sample DNA and said DNA in said compositionspecifically indicates the presence of said Nm or Ng in said sample,wherein said DNA in said composition is specific to Nm and Ng and doesnot hybridize on a Southern blot to a DNA sequence of Neisserialactamica (Nl) strain Nl8064 under the following hybridizationconditions: 16 h at 65° C., with a solution comprising 0.5 M NaPO₄ pH7.2, 0.001 M EDTA-Na, 1% bovine serum albumin and 7% sodiumdodecylsulphate, followed by at least two washes in a solutioncomprising 40 mM Na PO₄ pH 7.2, 1 mM EDTA, and 1% SDS, the final washbeing conducted at 65° C. for 5 minutes, and wherein said DNA is saidcomposition specifically hybridizes to Nm DNA and Ng DNA in the presenceof Nl DNA.